The pharmacological activities of all beta-adrenergic receptor agonists have one feature in common as they all activate adrenergic beta-receptors. Activation of adrenergic beta-receptors leads to increased intracellular concentration of cyclic adenosine monophosphate (cAMP), which triggers various events in various cells and organs. Cellular responses to beta-receptor activation include for example lipolytic activity in adipose tissues, smooth muscle relaxant activity of bronchial smooth muscle and increased frequency of contractions in the heart (Goodman-Gilman, The Pharmacological Basis of Therapeutics.) Most adrenergic beta-receptor agonists have affinity for two types of adrenergic beta-receptors. Thus, both salbutamol and ractopamine have affinity for adrenergic beta-1 and beta-2 receptors, but negligible affinity for beta-3 receptors (Example 2). There is no significant effect of ractopamine on adrenergic alpha-receptors according to Colbert et al., 1991, which publication is hereby included in its entirety by reference.
Adrenergic beta-agonistic drugs characteristically contain as part of their chemical structure an ethanolamine or 2-amino-ethanol moiety. Since the chemical structures of these drugs usually comprise at least one asymmetric carbon atom, these drugs commonly exist in optically active isomeric form, with the chiral carbon atom having (R) or (S) configuration. When there is one single asymmetric carbon atom present, the beta-receptor agonists exist as individual (R) or (S) enantiomers or in racemic (RS) form, i.e. as an approximately 50:50 mixture of (R) and (S) enantiomers. Compounds with two chiral centers—such as ractopamine—have four isomers, which are the RR-, SS-, RS-, and SR-isomers. For the sake of simplicity, RR-ractopamine may herein be referred to as RR or (RR), SS-ractopamine may herein be referred to as SS or (SS), RS-ractopamine may herein be referred to as RS or (RS), and SR-ractopamine may herein be referred to as SR or (SR). Compounds with four isomers (e.g. ractopamine) may exist in a number of forms i.e. in the single, pure RR or SS or RS or SR isomeric forms, or as mixtures of the compositions RR/SS, RR/SR, RR/RS or RS/SR, SR/SS or RS/SS. The compound ractopamine is a mixture of all four isomers. The term “optically pure isomer” or the like, as used herein, refers to a compound that contains at least 95% by weight of one isomer while the total concentration (i.e. the sum) of the corresponding and remaining isomers is 5% or less by weight, based on the total amount of ractopamine present.
RR/SS/RS/SR-ractopamine is a mixture of all four isomers in approximately similar concentrations. All four isomers usually exist in approximately the same concentrations of is approximately 25%. However, for the present purpose, RR/SS/RS/SR-ractopamine may contain from 23% to 27% of any of the four isomers.
Ractopamine has the molecular formula C18H23NO3 and is typically prepared as a hydrochloride salt. Chemically, ractopamine differs from dobutamine in the location of only one hydroxyl group, but ractopamine is not a catecholamine and is therefore not instantaneously metabolised by catechol-O-methyl transferase. Ractopamine HCl (4-hydroxy-a-[[[3-(4-hydroxyphenyl)-1-methylpropyl]amino]meth yl]benzenemethanol hydrochloride) has a molecular weight of 337.85 and a molecular formula of C18H23NO3.HCl (CAS number: 90274-24-1). The term ractopamine HCl refers to the hydrochloride salt of RR/SS/RS/SR-ractopamine. Thus, Ractopamine HCl (or ractopamine HCl) is the hydrochloride salt of a mixture of all four isomers in approximately equal proportions, as defined above.
The structure below depicts ractopamine. The two chiral centers (sites) are marked with asterisks (*). In order to differentiate between the two chiral centers (sites), they are here being called the “OH-site”, which is the benzylic stereocenter, and the “Me-site” (FIG. 1). Thus RR-ractopamine has the R-configuration at both sites, while SR-ractopamine has the S-configuration at the “OH-site” and the R-configuration at the “Me-site”.
FIG. 1. Chemical structure of ractopamine HCl; Isomers “OH-site”“benzylicChiral sitessite”“Me-site”Isomers:RRRRSRSRSSSSRSRS
Ractopamine is commercially available under the trade names PAYLEAN®, Elanco and OPTAFLEX®, Elanco and both are used as growth promotants for livestock.
Although structurally identical, isomers can have different effects in biological systems: one isomer may have specific therapeutic activity while another isomer may have no therapeutic activity or may have entirely different forms of biological activity. Of the four isomers of ractopamine, it is known that RR-ractopamine is the most potent, both when tested in vitro (Mills et al., 2003a) and in vivo (Ricke et al., 1999); both publications are hereby included in their entirety by reference. Thus, when tested for binding affinity for porcine adrenergic β-2 receptors, RR-ractopamine was about 2.5 times as active as the mixture of all four isomers (Mills et al., 2003a.)
The HCl salt of the RR-isomer of ractopamine is called Butopamine Hydrochloride, USAN and has been tested as a cardiac stimulator for humans (Thompson et al., 1980), which publication is hereby included in its entirety by reference. Butopamine is considered to be a full agonist at the beta-2-receptor sites (Smith, 1998; Mills, 2002; Mills et al., 2003a, 2003b). which publications are hereby included in their entirety by reference.
The relative contributions of adrenergic beta-1- and beta-2-receptor activation to the pharmacological effects of ractopamine may also differ by the different ratio of the beta-receptor subtypes in tissues and species. Beta-1- and beta-2-receptor are co-expressed in most tissues, but the ratio of these receptor subtypes can vary such that beta-1-receptors are predominant in heart (70-80% in humans, 72% in pigs) and adipose tissue (75% in rats, 80% in pigs), while beta-2-receptors are predominant in skeletal muscle (60% in pigs), uterus (80% in humans) and lung (65% in pigs, 80% in humans or horses) (Ungemach, 2004, which publication is hereby including its entirety by reference.)
The development of RR-ractopamine (butopamine) was discontinued, reportedly due to cardiovascular side effects, such as for example severe tachycardia (Thompson et al., 1980). Cardiac side effects, such as tachycardia, are also seen with isoprenaline, which is also a full agonist on cardiac beta receptors. RR-ractopamine has been found also to be a full cardiac agonist on cardiac beta receptors, while SR-ractopamine is a partial agonist. Thus, RR/SR-ractopamine has partial agonistic activities. Full cardiac agonistic activity by an adrenergic beta-receptor agonist implies that said compound has adrenergic beta-receptor stimulating activity, while being devoid of adrenergic beta-receptor blocking activity. It is concluded that by using RR/SR-ractopamine instead of RR-ractopamine, the risk for cardiac side effects is decreased.
Adrenergic beta-receptor agonist drugs can have pharmacological and toxicological side effects that range from minor importance to major importance. Bronchial smooth muscle relaxation by adrenergic beta-2 stimulation may be a side effect of minor importance for healthy livestock animals. Ractopamine has been found to cause increased heart rate and CNS-mediated stress in livestock animals (Marchant-Forde et al., 2003, which publication is hereby included in its entirety by reference.) These are side effects of major importance, particularly since ractopamine is increasing the stress levels in animals—even during times with increased basal stress for the animals, such as during handling and (Marhant-Forde et al., 2003). Stress in swine, may induce the PSE syndrome in the animals, which means poor meat quality that is pale, soft and exudative, and becoming dry upon cooking.
As mentioned above, ractopamine is known to cause tachycardia in livestock animals, while R-salbutamol has the advantage of not causing tachycardia in the livestock animals. In the case of ractopamine, it has been suggested that the significant tachycardia in livestock animals may in part be caused by CNS-mediated stress (Marchant-Forde J. N., et al., 2003 and London C. J., et al, 2005, which publications are hereby included in their entirety by reference.) The combination of stress-induced tachycardia and beta-receptor mediated tachycardia is a serious side-effect of ractopamine and leads to cardiac tachyarrhythmias and increased lethality of livestock animals by sudden cardiac death (cardiac ventricular fibrillation.)
In many animals including livestock animals, stress manifests itself—directly or indirectly—in a range of forms extending from irritability to aggression. As pointed out above, stress may lead to cardiovascular side effects ranging from slightly elevated heart rate to serious tachycardia and cardiac arrhythmias, which in turn can lead to sudden death. The prevalence of stress-induced lethality varies among species; some having higher stress responsiveness than others (Odeh et al., 2003, which publication is hereby included in its entirety by reference.)
Stress in horses can be expressed in various ways, such as for example nervousness, anxiety and tachycardia and can be caused for example by heat, transportation and feed withdrawal. Stress in horses can also be induced by drugs or aggravated by drugs, such as for example adrenergic beta-receptor agonists that may be given to the horses of various reasons, such as for example as bronchodilators in heaves. CNS-mediated stress in horses may also lead to increased susceptibility for various diseases, such as for example allergic diseases or infectious diseases such as opportunistic bacterial infections. The use of an adrenergic beta-agonist that does not induce stress is particularly important in animals that are already suffering from stress or have a propensity for developing stress.
Stress in pigs is very common and some pigs have been shown to carry a specific stress-gene. Pigs that are homozygous to this gene are particularly stress-prone although heterozygous pigs are also more stress-prone than pigs that do not at all carry or express the stress-gene (Sterle, 2005, which publication is hereby included in its entirety by reference.) CNS-mediated stress in pigs can be expressed in various ways, such as for example aggression, tail-biting, and tachycardia and can be caused for example by heat, transportation, stocking density, human interventions, feed withdrawal, disease and aggression between males. Stress in pigs can also be caused or aggravated by drugs, such as for example ractopamine (Marchant-Forde et al. 2003.) Porcine Stress Syndrome (PSS) is triggered when pigs are subjected to stress associated with transportation, restraint, fighting, mating, exercise or hot and humid weather. Pigs with PSS become dyspneic, hyperthermic, cyanotic, develop muscle rigidity and such animals often die prematurely. Some degree of stress can be observed in most pigs and most pigs may therefore have propensity for stress. The administration of certain drugs, such as ractopamine to pigs may induce or aggravate PSS in swine. In addition to the well-known fact that stress induces increased mortality in swine, it has been demonstrated that stress has a negative effect on the quality of meat (Purdue; http://ag.ansc.purdue.edu/meat quality/mqf stress.html.) Thus, the muscles from stress-positive pigs often show the PSE syndrome (pale, soft and exudative). This condition causes the carcasses to be classified as being of unacceptable or inferior quality, since the meat from such animals tend to become dry when cooked, (Stadler K., which publication is hereby included in its entirety by reference.) The use of an adrenergic beta-agonist that does not cause stress is particularly important in animals that are already suffering from stress or have a propensity for developing stress.
Stress in ruminants can be expressed in various ways and in cattle ranging from anxiety to aggression or depression, increased body temperature and increased heart rate. Stress in cattle can be caused by a variety of factors, such as changes in environment, transportation, human contact, aggressive herd behaviour and changes in the herd social rankings, hunger, thirst, fatigue, injury or thermal extremes. The propensity for stress in cattle seems to affect most animals and the administration of drugs, such as ractopamine may induce or worsen CNS-mediated stress in cattle and particularly in cattle that are predisposed for stress. Stress in cattle is a serious condition and may lead to decreased quality of the meat and increased lethality among the animals. The use of an adrenergic beta-agonist that does not cause stress is particularly important in animals that are already suffering from stress or have a propensity for developing stress.
As other examples of ruminants, sheep also develop symptoms of CNS-mediated stress due to the same or similar factors as described above for other species and may include but are not limited to changes in the environment, transportation, human contact, aggressive herd behaviour, hunger, thirst, fatigue, injury or thermal extremes. The symptoms of CNS-mediated (psychological) stress are similar to those of other species and include anxiety, aggression, increased body temperature or increased heart rate. The consequences of stress are similar to those described above for other species and include risk for decreased quality of meat and sudden death of the animals. The administration of drugs, such as ractopamine, may induce stress in sheep or increase the symptoms of stress in said species. Stress in sheep can be a serious condition and may lead to decreased quality of the meat and increased lethality among the animals. The use of an adrenergic beta-agonist that does not cause stress is particularly important in animals that are already suffering from stress or have a propensity for developing stress.
As still another example, birds such as chickens, ducks, geese, turkeys, ostriches, emus or quails may also develop CNS-mediated stress by doses of ractopamine, corresponding to those necessary for obtaining increased muscle weight, decreased fat deposits and improved feed efficiency. Particularly, chickens in “grower houses” are suffering from stress or are predisposed to stress because of the high stocking density (up to 20,000 birds or more in very confined space). Symptoms of stress in birds, such as for example chickens, ducks, geese, turkeys, ostriches, emus and quails, can be expressed in various ways, as for example, anxiety, aggression, increased body temperature, tachycardia and lethality and can be caused for example by heat, transportation, high stocking density, sudden environmental factors, feed withdrawal, injury or disease. The administration of the beta-receptor agonist ractopamine may induce or increase stress in birds. CNS-mediated stress in birds—and particularly in chicken—may lead to decreased quality of the meat and increased lethality among the animals.
Stress may also manifest itself in farmed fish, such as for example barramundi, carp, cod, perch, salmon, trout and tilapia. Symptoms of stress and symptoms for predisposition (propensity) for stress in farmed fish can be observed as increased activity as for example during feeding frenzy and stress can lead to sudden death of the fish. Stress in fish can be caused for example by extreme temperatures, environmental factors, disease, parasites, handling or transportation. The administration of exogenous beta-receptor agonists may lead to stress in animals that are predisposed for developing stress or may cause a worsening of the symptoms of stress in fish, leading to decreased quality of the meat and increased lethality among the animals. The use of an adrenergic beta-agonist that does not cause stress is particularly important in animals that are already suffering from stress or have a propensity for developing stress.
Stress in dogs and in cats may be manifested by vocalization, changes in appetite, aggressive behaviour or otherwise changed attitudes or behaviours, or in other ways, such as described for other species above. The administration of drugs, such as ractopamine may induce stress in all dogs and cats and also in dogs and cats that are used as companion animals. Stress in dogs and cats may happen particularly in predisposed animals, such as for example in certain strains of dogs. Stress in dogs and cats can be a serious condition and may lead sickness and increased lethality. The use of an adrenergic beta-agonist that does not cause stress is particularly important in animals that are already suffering from stress or have a propensity for developing stress. Beta-receptor agonists, such as for example RR/SR-ractopamine may be used in dogs and cats that are over-weight or are in need of exercise. Due to their pharmacological effects, certain adrenergic beta-receptor agonists, such as for example RS- or R-salbutamol and RR/SR-ractopamine can also be used in animals that are compromised by various diseases, such as for example heart failure, where these drug may be used alone or in combination with diuretics or other drugs as known by those skilled in veterinary medicine.
Stress in animals can be monitored, judged and rated by individuals who are skilled in the art of animal psychology. In addition to monitoring and rating the behaviour of the animals, objective parameters are being used, such as for example determination of the concentration of circulating corticosteroid levels. (Post et al., 2003, which publication is hereby included in its entirety by reference.) Depending on the species, stress in animals in response to exogenous adrenergic stimulation can also be monitored by parameters such as body temperature, heart rate, spontaneous motility, aggression, ease of handling and even weight loss (Marchant-Forde et al, 2003.)
The use of an adrenergic beta-agonist that does not cause stress is particularly important in animals that are already suffering from stress or have a propensity for developing stress. As mentioned above, predisposition of stress in livestock animals is common and it will be advantageous to avoid the worsening of the stress in these animals that is induced by ractopamine. As pointed out below, stress has now been found not to be caused by RR/SR ractopamine, which makes this mixture of ractopamine isomers particularly useful in all the various animal species mentioned above.